Neuropeptide release characteristically requires bursts of electrical activity and controls mood, behavior and development. We have used in vivo imaging of fluorescent neuropeptide-containing vesicles in fruit fly nerve terminals to study activity-dependent neuropeptide release and the regulated vesicle motion that sustains this release. Furthermore, optical experiments showed how vesicles generated in the neuronal cell body (also called the soma) are delivered to distant release sites based on activity. Here we extend these studies to address three key questions: 1. How is the activity dependence of neuropeptide vesicle mobility determined by Ca2+ signaling? 2. What is the role of cAMP signaling in activity-dependent neuropeptide release? 3. How are neuropeptide vesicles distributed among multiple release sites in a single neuron? Given the role of neuropeptides in pain, appetite, mood, and sleep, basic mechanisms underlying neuropeptide release could be clinically relevant. Furthermore, these studies are focused on maintenance of motor nerve terminal function by axonal transport, which is relevant to Amyotrophic Lateral Sclerosis (ALS) and other neurodegenerative diseases. Finally, experiments on neuronal insulin release will yield insights into the evolution of neuroendocrine control of growth and aging. PUBLIC HEALTH RELEVANCE: This project will elucidate mechanisms that regulate neuropeptide release, neuronal cell biology and aging. These mechanisms are key to understanding wide ranging brain functions including control of pain, sleep and mood, the maintenance of nerve terminal function that is important in neurodegenerative diseases and the evolution of neuroendocrine control of growth and lifespan.